Plant Science

AbstractThe bacterium Candidatus Liberibacter asiaticus (Las) has been strongly associated with huanglongbing, or citrus greening, which is one of the most devastating citrus diseases worldwide. Las is transmitted by the Asian citrus psyllid (ACP) Diaphorina citri (Hemiptera: Psyllidae) in a persistent manner, but its interactions with the psyllid vector, particularly at the organ and cellular levels, are poorly understood. We have tested several fluorescence in situ hybridization (FISH) protocols and three nucleic acid probes for the localization of Las in haemolymph smears and dissected organs of ACP adults that fed on Las‐infected plants in the field or laboratory and in sections from Las‐infected citrus leaves. Las was detected by FISH and confocal laser scanning microscopy in the filter chamber, midgut, Malpighian tubules, haemolymph, salivary glands, ovaries and in muscle and fat tissues of ACP that acquired Las from infected plants, as well as in the phloem of infected citrus leaves. Las appeared as pleiomorphic bodies or short thin rods that were much more dispersed and individually distinct in citrus leaf phloem and in ACP haemolymph, but more densely aggregated in cells of the salivary glands and other ACP organs and tissues. Our results provide the first in situ demonstration of Las infection in various psyllid organs and tissues and show the near‐systemic infection of ACP by Las.

The aim of this work was to assess the effects of a combined inoculum of a rhizobacterium and an arbuscular mycorrhizal (AM) fungus on plant responses to phytoplasma infection, and on phytoplasma multiplication and viability in Chrysanthemum carinatum plants infected by chrysanthemum yellows phytoplasma (CY). Combined inoculation with Glomus mosseae BEG12 and Pseudomonas putida S1Pf1Rif resulted in some resistance to phytoplasma infection (about 30%), delayed symptom expression in nonresistant plants, improved growth of the aerial part of the infected plants (+68·1%), and altered root morphology (root tip number: +49·9%; branching degree: +82·8%). Combined inoculation with the two beneficial microorganisms did not alter CY multiplication and viability. In inoculated and infected plants, phytoplasma morphology was typical of senescent cells. A more active and efficient root system in double‐inoculated plants probably mediated the effects of the two rhizospheric microorganisms in the infected plants. The practical application of rhizospheric microorganisms for mitigating phytoplasma damage, following evaluation under field conditions, represents an additional tool for the integrated management of phytoplasmosis.

Citrus Huanglongbing (HLB) has been threatening citrus production worldwide. In this study, a comparative proteomic approach was applied to understand the pathogenic process of HLB in affected sweet orange leaves. Using the isobaric tags for relative and absolute quantification (iTRAQ) technique, we identified 686 unique proteins in the mature leaves of both mock‐inoculated and diseased ‘Madam Vinous' sweet orange plants. Of the identified proteins, 20 and 10 were differentially expressed in leaves with and without symptoms of HLB (fold change > 2.5), respectively, compared with mock‐inoculated controls. Most significantly, upregulated proteins were involved in stress/defense response, such as four miraculin‐like proteins, chitinase, Cu/Zn superoxide dismutase and lipoxygenase. Microarray analysis also showed that stress‐related genes were significantly upregulated at the transcriptional level. For example, remarkable upregulations of miraculin‐like proteins and Cu/Zn superoxide dismutase transcripts were observed. Moreover, the transcriptional patterns of miraculin‐like protein 1 and Cu/Zn superoxide dismutase were examined at different stages of HLB disease development. Combined with the transcriptomic data, the proteomic data can provide an enhanced understanding of citrus stress/defense responses to HLB.

Winter oilseed rape (Brassica napus L.) is widely grown in Poland to produce vegetable oil for industrial processing of human and animal feed. In recent years, according to European Union directives on the use of biofuels (Directive 2003/30/EC), the area under oilseed rape cultivation in Poland has dramatically increased to 810,000 ha in 2009 and is still increasing. Morphological deformations of winter oilseed rape indicative of phytoplasma infection have been observed sporadically in Poland since 2000 (3). Plants exhibiting floral virescence, phyllody, as well as auxiliary bud proliferation, reduced leaves, and malformation of siliques were identified during surveys of research fields in Wielkopolska during May and June of 2009 and 2010. To confirm phytoplasma infection of these plants, inflorescence and leaf tissues were collected from nine diseased and three symptomless plants from three different field locations with 1 to 16% disease incidence. Total DNA was extracted from each plant tissue sample with a modified cetyltrimethylammoniumbromide method (2). Samples were analyzed for phytoplasma DNA with a nested PCR assay employing phytoplasma universal rRNA operon primer pair P1/P7 followed by R16F2n/R16R2, using previously described conditions (1). PCR products of 1.8 and 1.2 kb were obtained from all diseased plants only following PCRs with P1/P7 and nested primer pair R16F2n/R16R2, respectively. PCR products were not obtained from symptomless plants. Eight 1.2-kb amplicons were sequenced (GenBank Accession Nos. JN193475 to JN193482). Comparative analysis of the R16F2n/R16R2 rDNA sequences confirmed the phytoplasma origin of the rDNA sequences that shared 100 to 99% identity with Maize bushy stunt phytoplasma (GenBank Accession No. HQ530152), Alfalfa stunt phytoplasma (GenBank Accession No. GU289675), Primula green yellows phytoplasma (GenBank Accession No. HM590623), and other aster yellows group phytoplasmas. A 1.8-kb amplicon of isolate designated RzW14 was sequenced (GenBank Accession No. HM561990) and had 99% identity with Aster yellow group phytoplasmas from Lithuania (GenBank Accession Nos.GU223208 and AY744071). A virtual restriction fragment length polymorphism analysis of the 16S rDNA sequences from the R16F2n/R16R2 amplicons was performed with iPhyClassifier (4). Restriction profile comparisons identified all aster yellows group phytoplasmas as subgroup 16SrI-B strains. To our knowledge, this is the first report of a ‘Candidatus Phytoplasma asteris’-related strain infecting oilseed rape in Poland. References: (1) I. M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (2) A. C. Padovan et al. Aust. J. Grape Wine Res. 1:25, 1995. (3) M. Starzycki and E. Starzycka. Oilseed Crops 21:399, 2000. (4) Y. Zhao et al. Int. J. Syst. Evol. Microbiol. 59:2582, 2009.

In January 2011, tomato (Solanum lycopersicum) plants exhibiting stunting, yellow mosaic, short, chlorotic leaves, aborted flowers, and reduced-size fruits, symptoms similar to those exhibited by plants infected by ‘Candidatus Liberibacter solanacearum’ (2), were observed in approximately 5% of tomato plants in greenhouses in Jocotitlan in the State of Mexico, Mexico. Occasional plant recovery was also observed. Tomato plants in this facility were previously shown to be infected by Mexican papita viroid (MPVd), Pepino mosaic virus (PepMV), and aster yellows phytoplasma. Eight symptomatic leaf samples (designated MX11-01 to MX11-08) were collected and screened against selected tomato viruses and pospiviroids by reverse transcription (RT)-PCR using purified plant RNA or for ‘Ca. L. solanacearum’ by PCR using purified plant DNA. As expected, both PepMV and MPVd were detected in these samples. However, two ‘Ca. L. solanacearum’-specific PCR products (1,168 and 669 bp) were also amplified in two samples (MX11-02 and MX11-05) using primers OA2 (2) and OI2c (1) or CL514F/CL514R (3), respectively. Each ‘Ca. L. solanacearum’-specific PCR product was gel purified with Geneclean (Q-Biogene, Carlsbad, CA) and cloned into pCR2.1 using TOPO TA cloning kit (Invitrogen, Carlsbad, CA) and sequenced (Functional Biosciences, Madison, WI). Sequences of 16S rRNA (1,168 bp) in both isolates (GenBank Accession Nos. JF811596 and JF811597) were identical. However, the 669-bp 50S rRNA sequences in these two isolates (GenBank Accession Nos. JF811598 and JF811599) contained two single nucleotide polymorphism (SNP) mutations. BLASTn searches showed that both 16S rRNA and 50S gene sequences in MX11-05 were identical to the ‘Ca. L. solanacearum’ previously identified on potato in Chihuahua (GenBank Accession Nos. FJ829811 and FJ829812) and Saltillo (GenBank Accession Nos. FJ498806 or FJ498807) in eastern Mexico. These ‘Ca. L. solanacearum’ isolates were recently classified as the “b” haplotype (4). Alignment analysis of the ‘Ca. L. solanacearum' 16S rRNA sequences also revealed the conserved SNP mutations (g.212T > G and g.581T > C) in MX11-02 and MX11-05 as previously identified for other “b” haplotype isolates (4). ‘Ca. L. solanacearum’ was first identified in greenhouse tomatoes in 2008 in New Zealand (2). It has also been identified in greenhouse and field tomatoes in the United States. ‘Ca. L. solanacearum’ was previously reported to infect field tomatoes in Sinaloa, Mexico (3), which was recently considered as the “a” haplotype (4). To our knowledge, this is the first report of ‘Ca. L. solanacearum’ naturally infecting tomatoes in Jocotitlan in the State of Mexico, Mexico. The greenhouse tomato ‘Ca. L. solanacearum’ may be transmitted from infected solanaceous plants by potato psyllids (Bactericera cockerelli), which were observed in this facility. References: (1) S. Jagoueix et al. Int. J. Syst. Bacteriol. 44:379, 1994. (2) L. W. Liefting et al. Plant Dis. 93:208, 2009. (3) J. E. Munyaneza et al. Plant Dis. 93:1076, 2009 (4) W. R. Nelson et al. Eur. J. Plant Pathol. 130:5, 2011.